Protein Assay

What is the purpose of protein assay and what is important to keep in mind

Protein quantification is often needed before further isolation and characterization of a protein sample

• you need to know how much protein is in a sample before you can identify and isolate it

• Western blot

• Blood plasma and food samples

Protein assays do not discriminate between different proteins in a sample

• only protein concentration

• Also, the best assay to use depends on the situation

What factors are important when choosing what protein assay to use

Sensitivity: How little of a substance (analyte) the method can detect

Specificity: How good the assay is in discriminating between desired analyte and any interfering substances

How does the protein assay technique of UV light absorbance work

Beer-Lambert Law

• Using spectrophotometer,

• Light is passed through a solution, where some light is absorbed

• Path length (length of container) is kept constant, so light absorbed at a specific wavelength is proportional to the protein concentration

What is Beer-Lambert’s Law

What wavelength do proteins absorb light and why

At 280 nm (UV range)

• Aromatic amino acids have strong UV-light absorption

• Tyrosine

• Tryptophan

• Phenylalanine

What instrument is used to take the absorbance of a solution

A NanoDrop-Micro-volume spectrophotometer

What are the advantages and disadvantages of UV light adsorption assay

Advantages

• Simple and fast

• Does not require reagents

• Very sensitive (detection limit: 1 microg/mL)

Disadvantages

• Not ideal for protein mixtures

• Different proteins may vary in proportion of Aromatic AA

• Requires purely protein sample

• Non-protein content (i.e. DNA) can also absorb UV light

What are Biuret-based assay methods

Assays based on redox rxns

• The color of the solution is based on the reductive chelation (binding of metal atom) of cupric ions (Cu2+) and the biuret reagent under alkaline conditions

• Detects peptide bonds

What is in a Biuret reagent and what color is it under normal conditions

• Biuret molecule: formed when urea is heated and 2 urea are bonded together

• CuSO4 (provides Cu2+)

• NaOH (alkaline conditions)

• Potassium Sodium Tartrate (tartrate forms complex w/ Cu2+ ions)

It is blue under normal conditions

How does the biuret rxn work and what wavelength can absorbance be read at

In alkaline solution, cupric salts get reduced to cuprous ions

• then they form coordinated complexes w/ peptides containing 2 or more amide bonds and the biuret molecules

• Produces purple color

• Absorbance read at 540 nm

What is the disadvantage biuret-based assay

They are incompatible with reducing or chelating agents

What are the extensions of Biuret assay methods and why are they relevant

Lowry assay and Bicinchoninic acid assay (BCA)

• To amplify the signal and improve sensibility

What is a Lowry assay and what wavelength can absorbance can it be read at

A Biuret-based assay with 2 consecutive rxns to get a final blue color

• Rxn 1: Biuret rxn with Cupric ions

• Rxn 2: Reduction of FCR by peptide-bound cuprous ions

Read at 750 nm

What are the advantages and disadvantages of Lowry assay

Advantages

• Sensitive w/ broad working range ~1-2000 microg/mL

Disadvantages

• Time consuming, longer incubation times

• Sensitive to many substances like detergents (>1% SDS, bc of membrane proteins extractions require SDS and copper chelating reagents (EDTA needed to prevent protein degradation)

What is Bicinchoninic Acid Assay (BCA) and what wavelength can the absorption be read at

A Biuret-based assay with two consecutive rxns

• Rxn 1: Biuret rxn

• Rxn 2: BCA reacts directly with C1+ to produce a purple product under alkaline conditions

[Cu+ - 2BSA] purple product measured at 562 nm

What are the advantages and disadvantages of Bicinchoninic Acid Assay (BCA)

Advantages

• Highly sensitive

• Applicable on a broad range of protein concentrations

• Color complex is stable

• Lower susceptibility to detergents

• Provides better protein-to-protein uniformity (less affected by differences in protein composition)

Disadvantages

• Reducing agents (e,g, DTT) and substances that reduce copper also produce color in BCA assay

• Substances that chelate copper reduce amount of color produced with protein (interferes with accuracy)

• Rxn influenced by certain amino acids (cysteine, tyrosine, and tryptophan) which can produce color

What are dye-based method assays

Assays with dyes that change color upon binding to proteins

What is Bradford Assay and what wavelength can its absorbance be read at

A dye-based assay

• relies on interaction between basic/aromatic amino acid residues (particularly arginine) w/ Coomassie brilliant blue G-250 dye (CBB)

• Binding of CBB to proteins causes spectral shift from reddish-brown to blue

• Absorbance read at 595 nm

What are the advantages and disadvantages of Bradford Assay

Advantages

• Easy and quick procedure

• Abs measured at 595 nm (visible range)

• Extremely sensitive

• Mostly no interference from reagents in your sample (not affected by EDTA and reducing agents)

Disadvantages

• Generally produces less linear curves

• Short linear range means dilution of samples often needed

• Concentration can only be accurately measured on a linear range of a curve

• Incompatible with detergents

• Low protein-to-protein variation, cannot bind to protein as effectively if it has low # of arginine/aromatic residues (underestimates protein concentration)

What assay should you use for samples that contain detergents

Copper-based assays such as BCA

What assay should you use for samples that contain reducing agents (like DTT)

Bradford assay

What assay should you use for samples containing EDTA

• EDTA chelates copper

• Use Bradford assay